Erythropoietin is a prime regulator of red cell development. Substantial progress has been made in studying its structure. However, very little is known about its mode of action, especially on the molecular level. The proposed research is designed to explore the action of pure, recombinant erythropoietin on the continuous erythropoietin-responsive cell line Rauscher murine erythroleukemia. We will examine the mechanism by which the signal resulting from the erythropoietin-receptor interaction is transmitted to the interior of the cell. Recent evidence indicates that erythropoietin rapidly alters the phosphorylation of one or more proteins in the erythroid cell membrane. These erythropoietin-sensitive phosphoproteins (ESPPs) may serve as second messengers for the hormone's action. This study will focus on the characterization of these ESPPs and on the elucidation of their functions and role in controlling erythroid differentiation. The ESPPs will be identified and characterized. This includes isolation and purification, protein sequence analyses, and isolating and sequencing corresponding cDNA clones. Their subcellular distribution will be examined using specific antibodies, and alterations in this distribution accompanying erythropoietin induction will be explored. The presence and distribution of these ESPPs in cells that are nonresponsive to erythropoietin will be assessed also. Radiolabeled cDNA probes will be employed to quantify changes in ESPP expression during erythropoiesis and to determine if this might serve as a controlling factor. The protein kinases involved in these phosphorylation reactions will be investigated and their relationship to erythropoietin and to its receptor will be determined. This research program represents the beginning of a systematic study of erythropoietin's action on a molecular level. The knowledge gained will be integral to our understanding of normal erythropoiesis and should prove relevant to the pathophysiology of disorders of red cell production.